Tai-Yuan Chen , Zhi-Chyang Kang , Ming-Tsung Yen , Ming-Hsing Huang , Bor-Sen Wang
This study investigated the inhibitory effects of aqueous extracts from Miracle Fruit leaves (AML) on mutation and oxidative damage. The results showed that AML in the range of 1–5 mg/plate inhibited the mutagenicity of 2-aminoanthracene (2-AA), an indirect mutagen, and 4-nitroquinoline-N-oxide (4-NQO), a direct mutagen toward Salmonella typhimurium TA 98 and TA 100. On the other hand, AML in the range of 0.05–0.2 mg/ml showed radical scavenging, reducing activities, liposome protection as well as decreased tert-butyl hydroperoxide (t-BHP) induced oxidative cytotoxicity in HepG2 cells. High performance liquid chromatography (HPLC) analysis suggested that the active phenolic constituents in AML are p-hydroxybenzoic acid, vanillic acid, syringic acid, trans-p-coumaric acid and veratric acid. These active phenolic components may contribute to the biological protection effects of AML in different models. The data suggest that AML exhibiting biological activities can be applied to anti mutation as well as anti-oxidative damage.
Introduction
The production of DNA damage plays an important role on mutation and ageing diseases. Various mutagens present on food increase oxidative stress and cancer risk through different mechanisms in cells. The metabolic products of these mutagens chieflfly bind to DNA at guanine residues. The consequence is that whenever these DNA adducts is generated, DNA mutation may increase, eventually increasing the risk of tumour progression. Except DNA damage, these mutagens may be metabolized and induce harmful oxidative stress, which also destroy the biological molecules (e.g. lipids) and cause mutations.
However, many reports suggest that intracellular oxidative stress derived from reactive nitrogen species (RNS) and reactive oxygen species (ROS) arises during physiological metabolism and after exposure to various chemical encouragements. Therefore, t-BHP is often used as a model compound for inducing oxidative stress during in vitro and in vivo studies. Further, oxidative stress can be observed in different pathological states, such as atherosclerosis and cancer. Therefore, the inhibition of oxidative stress may play an important step in preventing mutation and ageing diseases.
On the other hand, to decrease the oxidation of lipids, various antioxidants have been used to protect the lipids in foods from oxidation. However, because of safety concerns, there is currently an interest in replacing synthetic antioxidants with natural antioxidants. Thus, investigations on the natural inhibitors of oxidation in foods have received much attention. The Miracle Fruit shrub, Synsepalum dulifificum Daniell, bears red berries which contain a taste-modifying protein, miraculin.
To our knowledge, our study is the first to examine the biological effects of Miracle Fruit leaves, an agricultural waste material, on mutation and oxidation. The objective of this work is to determine the antimutagenic and anti-oxidative activities of aqueous extract from Miracle Fruit leaves.
Materials and Methods
2.1. Materials
4-Nitroquinoline-N-oxide (4-NQO), 2-aminoanthracene (2-AA), thiobarbituric acid (TBA) and tert-butyl hydroperoxide (t-BHP), 2 0 ,7 0 -Dichlorodihydroflfluorescein diacetate (DCF-DA) and chloromethylfluorescein-diacetate (CMF-DA), Culture medium and top agar ,Miracle Fruit leaves.
2.2. Sample preparation
The Miracle Fruit leaves were grounded after freeze-drying. The powder (10 g) was extracted with water (200 ml) at 100 C for 30 min and then centrifuged at 10,000g for 20 min. The extract was filtered and the residue was re-extracted under the same conditions. The combined filtrate was then freeze-dried. The yield obtained was 8.86% (w/w). The final sample was named as AML (the aqueous extract of Miracle Fruit leaves).
2.3. DPPH radical inhibition assay
The effect of samples on the DPPH radical was in the samples (0.1–0.4 mg/ml, 1 ml) were added to a methanolic solution (1 ml) of DPPH radical (final concentration of DPPH was 0.2 mmol/l). The mixture was shaken vigorously and allowed to stand at room temperature for 30 min; the absorbance of the resulting solution was then measured at 517 nm.
2.4. Reducing activity assay
The reducing power of AML was determined in the sample (0.1–0.4 mg/ml, 2.5 ml) were added to potassium ferricyanide (2.5 ml, 10 mg/ml), and the mixture was incubated at 50 C for 20 min. Trichloroacetic acid (2.5 ml, 100 mg/ml) was added to the mixture, which was then centrifuged at 650g for 10 min. The supernatant (2.5 ml) was mixed with distilled water (2.5 ml) and ferric chloride (0.5 ml, 1.0 mg/ml), and then the absorbance was read at 700 nm. The reducing activity was calculated against an ascorbic acid calibration curve.
2.5. Liposome oxidation assay
A solution containing the lecithin (500 mg) and phosphate buffer (50 ml, 10 mM, pH 7.4) was sonicated by an ultrasonic cleaner (Branson 8210, Branson Ultrasonic Corporation, Danbury, CT, USA) in an ice-cold water bath for 2 h. The sonicated solution (1 ml), FeCl3 (0.12 mM, 1 ml), ascorbic acid (0.5 mM, 1 ml) and AML (0.2–0.8 mg/ml, 1 ml) were mixed and incubated at 37 C for 1 h. The levels of liposome oxidation were determined.
2.6. Measurement of HepG2 cells viability
HepG2 cells (ATCC No. CRL-11997) were purchased from Bioresources Collection and Research Center (Shin-chu, Taiwan) and cultured in minimum essential medium (MEM) containing 10% fetal bovine serum and maintained in humidified 5% CO2/95% air at 37 C. After cells were cultured with AML (final concentration was 0.05–0.2 mg/ml), in the presence of 0.2 mM t-BHP or not for 6 h, cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
2.7. Evaluation of reactive oxygen species (ROS) and glutathione (GSH) in HepG2 cells
DCF-DA was used to determine the generation of ROS in HepG2 cells. The cell-permeant 2 0 ,7 0 -dichlorodihydrofluorescein diacetate (DCF-DA) is a chemically reduced form of fluorescein used as an intracellular indicator for reactive oxygen species (ROS) production in cells. Upon cleavage of the acetate groups by intracellular esterases the non fluorescent DCF-DA is converted to DCFH that need an oxidation via H2O2 and a peroxidase to be transform in the highly fluorescent 2 0 ,7 0 -dichlorofluorescein (DCF). HepG2 cells were pretreated with DCF-DA (50 lM) for 30 min, and then AML (final concentration was 0.05–0.2 mg/ml) was added to the medium in the presence of 0.2 mM t-BHP or not. After incubation at 37 C for 3 h, the adherent cells were trypsinized and washed with normal saline. ROS produced from intracellular stress was detected using a Bio-Tek FLx800 microplate fluorescence reader (Winooski, VT, USA) with excitation and emission wavelengths of 485 and 530 nm, respectively. On the other hand, intracellular GSH levels were determined after staining cells with chloromethylfluorescein-diacetate (CMF-DA). CMF-DA form GSH adducts in a reaction catalysed by glutathione-S-transferase. After conjugation with GSH, CMF-DA is hydrolysed to the fluorescent 5-chloromethylfluorescein by cellular esterase. HepG2 cells were treated with AML (final concentration was 0.05–0.2 mg/ml) in the presence of 0.2 mM t-BHP or not. After incubation at 37 C for 3 h, the adherent cells were trypsinized and washed with normal saline. Intracellular GSH was detected using a Bio-Tek FLx800 microplate fluorescence reader (Winooski, VT, USA) with excitation and emission wavelengths of 485 and 530 nm, respectively.
2.8. Mutagenicity assay
The mutagenicity of AML was tested according to the Ames test with a 20 min first incubation at 37 C. The histidine-requiring strains of Salmonella typhimurium TA 98 and TA 100 were obtained from Taiwan Agricultural Chemicals and Toxic Substances Research Institute (Taichung, Taiwan). The external metabolic activation system, S9 mix (Molecular Toxicology, Inc., Boone, NC, USA) was prepared from Sprague–Dawley male rats treated with Aroclor 1254. Samples (0.1 ml, 10–50 mg/ml corresponding to 1–5 mg/plate) were added to the overnight cultured S. typhimurium TA 98 or TA 100 (0.1 ml) and S9 mix (0.5 ml) or 0.1 M phosphate buffer (0.5 ml, pH 7.4) in place of the S9 mix. The entire mixture was incubated at 37 C for 20 min before molten top agar (2.0 ml) was added and then spread out in a Petri dish containing 20 ml of minimum agar. The mixture was counted after incubating at 37 C for 48 h. The toxic effects of AML on S. typhimurium TA 98 and TA 100 was determined as previously described.
2.9. Antimutagenic activity assay
The antimutagenic activity of AML was assayed according to the Ames method except for the addition of mutagen before incubation. The concentrations of mutagens were tested as in a previous study. The mutagens used were 4-NQO (0.5 kg/plate), a direct mutagen and 2-AA (2.5 lg/plate), which required S9 mix for metabolic activation. Mutagen (0.1 ml) was added to the mixture of a strain (TA 98 or TA 100), and samples were added with the S9 mix for 2-AA or with phosphate buffer (0.1 M, pH 7.4) for 4-NQO. The mutagenicity of each mutagen in the absence of samples is defined as 100%. The number of spontaneous revertants in the absence of mutagens and samples was used as reference. The inhibition (%) of mutagenicity of the sample was calculated as following: Inhibition (%) = {1 [(No. of his+ revertants with mutagen and sample No. of spontaneous revertant)/(No. of his + revertants with mutagen No. of spontaneous revertant)]} 100.
2.10. High performance liquid chromatography (HPLC) assay
HPLC was performed with a Hitachi Liquid Chromatograph (Hit achi Ltd., Tokyo, Japan), consisting of two model L-7100 pumps, and one model L-7455 photodiode array detector. Sample
(10 mg/ml) was filtered through a 0.45 lm filter and injected into the HPLC column. The injection volume was 20 ll and the flow rate was 0.8 ml/min. The separation temperature was 25 C. The column was a Mightysil RP-18 GP (5 lm, 250 4.6 mm I.D.; Kanto Corporation, Portland, OR, USA). The method involved the use of a binary gradient with mobile phases as previously described. The plot of the peak-area (y) vs. concentration (x, lg/ml), the regression equations of the three marker compounds and their correlation coefficients (r) were as follows: p-hydroxybenzoic acid, y = 0.1812x + 0.1414 (r 2 = 0.9994); vanillic acid, y = 0.1037x + 0.0983 (r 2 = 0.9992); syringic acid, y = 0.0477x + 0.03 (r 2 = 0.9995); trans-p-coumaric acid, y = 0.032x + 0.0091 (r 2 = 0.9991); and veratric acid, y = 0.0939x + 0.0665 (r 2 = 0.9991).
2.11. Statistical analysis
All data were presented as means ± standard deviations (SD). Each test was performed in triplicate. Statistical analysis involved use of the Statistical Analysis System software package (SAS Institute Inc.). Analysis of variance was performed by ANOVA procedures. Significant differences between means were determined by Duncan’s multiple range tests at a level of p < 0.05.
Results
The HPLC chromatographic analysis showed that bioactive phenolic components were presented in AML. These phenolic components have been identified as p-hydroxybenzoic acid, vanillic acid, syringic acid, trans-p-coumaric acid, and veratric acid by measuring their retention time and UV absorbance, in relation to standards.
The mutagenicity of AML was determined by comparing the ratio of induced revertants to spontaneous revertants in the plates. These observations indicated that AML did not increase the mutagenicity of S. typhimurium TA 98 and TA 100. Furthermore, the antimutagenicity of AML on 4-NQO and 2-AA induced mutation in S. typhimurium TA 98 and TA 100 was examined. AML displayed dose-dependent protection against 4-NQO induced mutagenicity in S. typhimurium TA 98 and TA 100, without S9 activation. AML at levels of 1–5 mg/plate showed 0–17% inhibition of 4-NQO induced mutagenicity in TA 98 and 2–58% inhibition in TA 100. The antimutagenicity of AML on 2-AA induced mutation in S. typhimurium TA 98 and TA 100, with S9 activation. AML at levels of 1–5 mg/plate showed 6–80% inhibition of 2-AA induced mutagenicity in TA 98 and 34–96% inhibition in TA 100. These observations indicated that AML could inhibit the mutagenicity of both direct and indirect mutagens in vitro.
Discussion
In this study, AML demonstrated multiple biological activities, including anti mutation and antioxidation. A considerable number of studies suggested that the effects of natural antioxidants, such as phenolic components of plant extract, in the biological systems provided protection because they scavenged radicals, chelated metals, and inhibited the oxidases and then regulated cellular redox state. Twelve phenolics were identified and quantified in the miracle berry flesh
Therefore, phenolic constitutes of AML such as p-hydroxybenzoic acid, vanillic acid, syringic acid, trans-p coumaric acid, and veratric acid were examined in this study. The phenolic constitutes of plant extracts could exhibit reactive species scavenger and regular oxidative stress.
In this study, the liposome was prepared from phospholipid and used as a lipid oxidation model to imitate the lipid oxidation of biomolecules. AML demonstrated a protective effect against the lipid damage caused by the hydroxyl radicals produced from a Fenton-like reaction.
In this study, the AML and its five marker components provided protection against lipid oxidation, indicating that AML could protect bio-lipid molecules from oxidative stress and prevent DNA damage in tissues. The equivalent concentrations of p-hydroxyben zoic acid, vanillic acid, syringic acid, trans-p-coumaric acid, and veratric acid in 0.2 mg/ml of AML were 0.0003, 0.0003, 0.0006, 0.0006 and 0.0002 mg/ml, respectively. The antioxidant potential depends on the substitution of the phenol ring with hydroxyl groups in ortho or para position. The methylation of the phenol ring in ortho position relative to the hydroxyl group also increased the activity. AML shows neither toxicity nor mutagenicity toward S. typhimurium TA 98 or TA 100 in the present study.
However, S. typhimurium is a bacterium and thus not a perfect model of the human body. In fact, some substances that cause cancer in laboratory animals do not give a positive Ames test. On the other hand, the bacterial reverse mutation test is rapid, inexpensive and relatively easy to perform. We determine the highest amount of test substance to be used by cytotoxicity test. In this study, AML (1–5 mg/plate) did not show any cytotoxicity against TA 98 or TA 100. Therefore, the recommended maximum test concentration for non-cytotoxic substances is 5 mg/plate.
These data implied that the conjugated reaction between AML and the toxic electrophile was an important detoxification pathway. On the other hand, AML might play an antimutagenic role by scavenging the active metabolic electrophile of 4-NQO and 2-AA. The anti mutagenic effects of the AML might also be attributable to decreased metabolic activation and the levels of toxic reactive intermediates, which further indirectly reduced cellular oxidative stress and, thereby, prevented mutation.
In summary, as mentioned above, AML demonstrates anti mutation and antioxidation effects. These activities may be partially attributable to its phenolic constituents. Furthermore, the five marker compounds have similar activities to the extract that contain significantly lower levels of the five marker compounds.
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